Project Summery / Abstract: The underlying hypothesis of this proposal, and one that has become the driving force of my research program, is that epigenetic and chromatin modifications are critical during cardiac development and will emerge as important therapeutic targets for cardiac diseases. While numerous individual genes that are regulated during cardiac development have been described, global transcriptional regulators and epigenetic modifiers of this process have been less well characterized. Histone deacetylases (HDACs) modify chromatin structure and affect local and global gene expression in the heart and elsewhere. Recently, I have discovered that global loss of Hdac2 in mice results in a partial perinatal lethality with severe developmental myocardial defects. Interestingly, Hdac2 affects the balance between differentiation and proliferation of cardiomyocytes. Previously, we have shown that Homeodomain only protein (Hopx) is expressed in the embryonic and adult heart and functions, at least in part, by directly interacting with Hdac2 to mediate the repression of myocardial genes. Global loss of Hopx in mice also results in a partial perinatal lethality and cardiac defects that resemble Hdac2 knockouts. Here, we show that Hdac2 and Hopx are co-expressed in the developing heart and loss of both Hdac2 and Hopx results in complete perinatal lethality with severe cardiac defects including muscular ventricular septal defects and markedly increased myocyte proliferation. Microarray analysis reveals dysregulation of several cell-cycle specific genes as well as cardiac structural genes in Hdac2- Hopx-null hearts. Our mechanistic analysis indicates that loss of both Hdac2 and Hopx leads to activation of Gata4, which has been shown previously to regulate myocyte proliferation. Hdac2 interacts with Gata4 and loss of Hdac2-Hopx increases Gata4 acetylation and activation in developing myocardium. These results suggest that the interaction between Hdac2 and Hopx is functional during cardiac development and therefore, I will test the hypothesis that Hdac2 and Hopx coordinately function in the heart to regulate Gata4 activity by directly regulating Gata4 acetylation and that this accounts for changes in myocyte proliferation. Specifically, I will investigate the mechanism by which Hdac2-Hopx complex regulates Gata4 acetylation and activity during myocyte proliferation and the effects of tissue specific loss of Hdac2-Hopx function in the developing myocardium. This will be accomplished by pursuing the following specific aims: Aim 1: Determine and characterize whether Hdac2 and Hopx function coordinately to regulate Gata4 acetylation and transcriptional activity in vitro and in vivo. A) Characterize the Hopx-Hdac2-Gata4 complex. B) Determine whether Hdac2-Hopx deacetylates Gata4. C) Determine and characterize whether Hdac2-Hopx regulates Gata4 transcriptional activity. Aim 2: Characterize the tissue specific role of Hdac2-Hopx complex in cardiac development through analysis of a newly generated floxed allele of Hdac2. PUBLIC HEALTH RELEVANCE: Project Narrative: Congenital heart defects are the most commonly occurring developmental defects in humans. Contributions of epigenetic and chromatin modifications to congenital heart diseases are largely unknown. The set of experiments outlined in this proposal have broad significance for understanding the fundamental mechanisms underlying myocyte proliferation and heart development.